Processing
NanoDrop instruments (Thermo Fisher Scientific) are commonly used to quantify nucleic acid molecules, including DNA oligonucleotides, double-stranded DNA, and RNA. Specifically, when measuring oligonucleotides, it is important to acknowledge their unique characteristics, which can require adjustment of NanoDrop parameters for correct readings.
Here, we provide a summary of recommended procedural adjustments, when using the NanoDrop instrument to quantify oligonucleotides.
1. Use an oligo-specific conversion factor instead of the general single-stranded DNA (ssDNA) conversion factor of 33 μg/A260.
Oligonucleotides are short, single-stranded molecules. Their UV spectrum and extinction coefficient are much more dependent on their base composition and sequence context compared to longer ssDNA and RNA molecules [1]. Thus, for quantification, IDT recommends that you determine and apply an oligo-specific conversion factor. The NanoDrop software will do this for you, when you choose either "custom" or "oligo" option from the sample type menu.
2. Use the MicroArray module of the NanoDrop software for measuring oligonucleotides with modifications.
Many of the modifications added to the 5′ or 3′ ends of oligonucleotides absorb light, and as a result, can affect quantification results [1]. Thus, for quantification of modified oligonucleotides, IDT recommends that you use a correction factor. To obtain this value with the NanoDrop software, use the MicroArray module, which provides an automatic correction for modification absorbance.
3. Make dilutions to ensure absorbance measurements use 1 or 0.2 mm path lengths.
Using multiple path lengths, NanoDrop instruments measure a wide concentration range of nucleic acids [2]. However, an instrument’s acceptable error increases as the path length is shortened [3]. The most precise quantification results can be obtained by measuring diluted oligonucleotides with an absorbance <12.5. This will help to ensure the instrument makes absorbance measurements at a 1 or 0.2 mm path length, providing a smaller error tolerance [3,4].
Determine the approximate absorbance of an oligonucleotide stock solution using the Beer-Lambert equation:
A = ε b c
Where:
A = Absorbance
ε = Molar attenuation coefficient (L/(mole·cm), obtained from the manufacturer)
b = Path length (cm)
c = Concentration (M, mole/L)
4. Turn off the default baseline correction for oligonucleotides with modifications that absorb light at 340 nm.
The default setting for NanoDrop instrument measurements is 340 nm. This baseline correction is necessary because it adjusts for any light scattering that may skew results. However, it is important to note that certain oligonucleotide modifications absorb light at this wavelength [1], therefore this baseline correction may also impact your results.
It is also noteworthy that traditional purity ratios (A260/280 and A260/230), used as an indication of the presence of various contaminants in nucleic acid samples, do not apply to oligonucleotides, because the shape of the oligonucleotide UV spectrum is highly dependent on base composition [1].
